A control system for gene expression is extremely important as basic means for each of protein production, metabolic engineering, and synthetic biology. Biotechnology that uses the control system for gene expression is employed in various fields such as mass production of a useful protein, metabolic engineering, and a whole cell biosensor. There is a demand for a technology for producing a control system for gene expression with desired properties, which may be naturally derived or may be artificially created, as required and rapidly.
In the natural world, there are various transcription and/or translation control mechanisms and sensor mechanisms. In recent years, a genetic switch has been reported as a control system for gene expression (Non Patent Literatures 1 to 5). The genetic switch is a molecular device for switching expression and non-expression (ON/OFF) of a specific gene with the use of any of various pieces of information as input. Through construction of a genetic circuit by integrating various genetic switches, it is becoming possible to construct an oscillator, a counter, a logic circuit, and the like in cells. In the genetic circuit to be constructed in cells, a plurality of genetic switch mechanisms have to work in cooperation to finally establish an integrated function. To that end, all the switches forming the circuit are required to work with certain respective properties. In order that an arbitrary genetic circuit can be freely designed, a tremendous number of genetic switches with different functions/properties are required.
For example, the genetic switch can be applied to mass production of a useful protein. The production of a useful protein frequently employs an approach involving forcing host cells such as Escherichia coli (hereinafter sometimes abbreviated as “E. coli”) to express a target protein obtained from an organism of a different species. However, there are many proteins showing toxicity to the host cells. In case of producing any such protein, the host cells are made to proliferate to a sufficient number, and then the forced expression is caused by “inducing” expression at appropriate timing. In this case, the following two conditions are required: (1) a basal expression level in an uninduced state is sufficiently low (i.e., stringency, less “leakiness”); and (2) sufficient gene expression is achieved when the expression is induced (ON) (i.e., a ratio between expression levels in ON/OFF states is large). In order to achieve the conditions, there have been developed various promoter systems such as the pET system (manufactured by Novagen). However, the search for an optimum genetic switch is still in progress.
The genetic switch can also be used as means for metabolic engineering. In metabolic engineering, a biosynthetic pathway of a given substance of interest is constructed by simultaneously expressing a plurality of enzyme genes in one host cell. In pursuing the best results in the constructed artificial biosynthetic pathway, such as a maximized yield of a final product per biomass and a minimized amount of a by-product, it is vital to regulate and investigate expression levels of individual genes meticulously, and if possible, independently. For this purpose, a large number of genetic switches with desired ON/OFF switching properties are required. Particularly in the case of simultaneously regulating expression of a plurality of genes in one cell, functions required of genetic switches include, for example the following: (1) genetic switches are mutually orthogonal, i.e., an inducer for one genetic switch does not cause improper operation of another genetic switch; and (2) an level of expression by each genetic switch can be continuously regulated.
The genetic switch can also be used as a biosensor. Cells detect large amounts of chemical information and/or physical information, and express an appropriate group of genes in response thereto. There has been developed a cell sensor (whole cell sensor) having a reporter gene such as a green fluorescent protein (GFP) ligated downstream of the “substance detection” system. A demand exists for development of a sensor for an arbitrary substance (or physical stimulation) through, for example, proper modification of the already developed sensor.
When the genetic switches and sensor systems as described above can be created in large numbers, complex genetic circuits provided with information integration and/or processing (assessment) functions can be created by combining the genetic switches and the sensor systems. However, the genetic switches included in the genetic circuit have problems of leakiness and cross-talk of switches, which correspond to leakage of electricity in an electronic circuit. Hence, when a plurality of genetic switches are simultaneously used, the genetic switches do not properly work. Regarding switching properties of genetic switches, such as an ON/OFF threshold and a dynamic range, when complex genetic circuits are designed by combining the genetic switches, it is strongly required to: (1) arbitrarily change a response threshold to cause an expression trigger; (2) repress “leakiness” of expression under an uninduced state; and (3) secure orthogonality to other factors in cells. The de novo design of those requirements into a complex genetic circuit is an extremely difficult task. Therefore, the limit of integration of a circuit in cell engineering is extremely low at present. In order to overcome those problems, improvement of the genetic switch has been demanded.
On the other hand, even a complex circuit, when seen as a whole, can be regarded as one genetic switch that stipulates a triggered state of a gene depending on input conditions. That is, the genetic circuit triggers expression of a downstream gene (set) under certain conditions, and represses the expression under other conditions.
Therefore, in construction of genetic circuits, through selection of those in a triggered state when gene expression should be ON and/or those in a repressed state in a situation where gene expression should be OFF (ON selection/OFF selection), it is possible to select and/or obtain genetic circuits having arbitrary output properties. When the ON selection/OFF selection can be easily and successively conducted, various genetic switches (or genetic circuits) can be rapidly developed. In functional selection of genetic switches, it is necessary to select genetic switches in both the ON state and the OFF state under various input conditions (Non Patent Literatures 6 to 12). That is, ligation of two selectors, i.e., an ON-selector and an OFF-selector to an output side, i.e., downstream of a genetic circuit enables the functional selection of a genetic switch or an integrated circuit thereof, i.e., a genetic circuit. In molecular genetics, various ON-selectors and OFF-selectors are known.
Recently, an attempt has been made to cause one gene to conduct the functions of the ON-selector and the OFF-selector (Non Patent Literatures 10 to 13). A selector having both the functions of the ON-selector and the OFF-selector is called a dual selector. An “operon-type” selection method involving using an independent ON-selector and OFF-selector has a problem in that genetic mutations frequently occur in one of the selector genes, resulting in the frequent emergence of false positives. However, the dual selector does not have such problem. Although there are few reports of dual selectors for the functional selection of genetic switches, there have been reported systems each using a gene that imparts antibiotic resistance to cells, such as a system using a tetracycline resistance gene tetA (Non Patent Literatures 10 and 11) and a system using a chloramphenicol resistance gene CAT (Non Patent Literature 12), and a system using a chemotaxis gene cheZ of E. coli (Non Patent Literature 13). The system using tetA conducts dual selection by utilizing a bactericidal mechanism of cells through control of the transcription of tetA and measuring the survival or death of cells. The system using cheZ conducts dual selection based on the presence or absence of mobility of cells through control of the translation of cheZ.
Hitherto, various selection systems for use in ON selection/OFF selection have been developed. However, each of the systems achieves selection by the so-called selective proliferation, in which cells can proliferate when a genetic circuit transfected into the cells is properly output. Such selection system includes a cell proliferation process in each selection operation, and hence requires about 12 hours to 24 hours for each selection operation. Therefore, selection of a genetic circuit, in particular, selection of a complex genetic circuit, requires a large number of days. As described above, the conventional approaches have the problem of requiring a long time period for a selection operation, or such problem that selection efficiency is affected by the selection conditions.